Scientists supported by the National Institute of Dental and Craniofacial Research (NIDCR), part of the National Institutes of Health, report that women who smoke during pregnancy and carry a fetus whose DNA lacks both copies of a gene involved in detoxifying cigarette smoke substantially increase their baby's chances of being born with a cleft lip and/or palate.

According to the scientists, about a quarter of babies of European ancestry and possibly up to 60 percent of those of Asian ancestry lack both copies of the gene called GSTT1. Based on their data, published in the January issue of the American Journal of Human Genetics, the scientists calculated that if a pregnant woman smokes 15 cigarettes or more per day, the chances of her GSTT1-lacking fetus developing a cleft increase nearly 20 fold. Globally, about 12 million women each year smoke through their pregnancies.

Dr. Jeff Murray, a scientist at the University of Iowa and senior author of the study, noted that parents who are considering having a child and need added motivation for the mother to quit smoking might one day be tested to determine their GSTT1 status. Because the fetus inherits its genes from both mother and father, the test would determine the likelihood of the baby developing without the GSTT1 gene to detoxify the cigarette smoke.

"A test that indicates the GSTT1 gene is present certainly would not eliminate a baby's risk of a cleft because many other genetic and environmental factors can be involved." said Murray. "But the opposite result would give the mother one more compelling reason to quit smoking for her own health and for the sake of her child."

In the United States, about one in every 750 babies is born with isolated, also called nonsyndromic, cleft lip and/or palate. The condition is correctable but typically requires several surgeries. Families often undergo tremendous emotional and economic hardship during the process, and children frequently require many other services, including complex dental care and speech therapy.

According to Murray, researchers have built a strong statistical case over the past several years that pregnant women who smoke put their unborn babies at greater risk of developing a cleft. The data raised two related questions. "Do genetic variations in the mother influence her own metabolism of the cigarette smoke and its byproducts, thus setting in motion developmental changes that cause the cleft in the fetus? Or do genetic variations in the fetus itself compromise its ability to metabolize the cigarette smoke and cause the cleft?" said Dr. Min Shi, now a scientist at NIH's National Institute of Environmental Health Sciences and a lead author of the paper.

To find the answers, Murray's group teamed with colleagues in Denmark to perform a large, complex, and possibly first-of-its-kind international study. The group first assembled a list of 16 genes of interest, each of which encode proteins that plug into various pathways in the body involved in detoxifying dangerous chemicals. "We picked genes that previous evidence shows either are directly involved in cigarette smoke toxicity or are major players in general toxicity management in people," said Dr. Kaare Christensen, a scientist at the University of Southern Denmark in Odense and an author on the paper.

"These genes tend to be quite variable from person to person in their precise DNA structure, or spelling," Christensen added. "We wanted to see if any of these variations might adversely affect a person's ability to break down the toxic products of cigarette smoke."

Christiansen and his colleagues then turned to their existing database of kids with clefts, their parents, and siblings. In all, the scientists analyzed 5,000 DNA samples from both continents — including 1,244 from children born with clefts. Importantly, the families in Denmark and Iowa provided the opportunity to independently confirm the findings in two distinct populations.

In addition, they had free public access to the NIDCR-funded COGENE project, a comprehensive online database of genes expressed throughout the various stages of development. Working closely with Dr. Mike Lovett at Washington University in St. Louis, one of COGENE's founders, the database proved especially helpful because cleft lip and/or palate occurs during the first 5-to-12 weeks of development. This meant the scientists had to be sure not only that their genes of interest are expressed during this vital period but are switched on in fetal craniofactial structures. If the genes met both criteria, the investigators said they hoped their subsequent data might point them to a gene-environment interaction.

As reported, the scientists determined from their analyses that the mother provides the toxic environmental exposure, which then can be greatly amplified by the genetics of the fetus to produce the cleft. This marks the first time a gene-environment interaction in clefting has been documented at a molecular level. The data also point the way for future studies to define the specific molecular chain of events that lead to the cleft, vital information to understand and hopefully one day prevent the process.

While sifting through the data, the researchers took particular note of the GSTT gene and its contribution to clefting. The gene encodes one of the body's approximately 20 different glutathione S-transferase enzymes. These enzymes collectively play roles in common detoxification processes, ranging from chemically altering drugs and industrial chemicals to detoxifying polycyclic aromatic hydrocarbons, a key component of cigarette smoke.

The scientists found that pregnant women who smoked and also carried fetuses that lacked the GSTT1 enzyme were much more likely to give birth to a baby with a cleft. This finding was true in Iowa and Denmark, and they noted in the COGENE database that the gene is highly expressed in developing craniofacial structures. "It may be that be that the lip and palate can form normally without GSTT1," said Murray. "But if the chemicals in cigarette smoke challenge the normal development of these structures, fetuses that lack the gene are at a distinct disadvantage."

Murray and his collaborators continue their genetic analyses. "We now have data from about 350 genes on this cohort of families," he said. "It's certainly a more complicated analysis to perform, but we're working our way through it and hope to have some very interesting data in the months ahead."

The article is titled "Orofacial Cleft Risk is Increased with Maternal Smoking and Specific Detoxification-Gene Variants," and is published in the January 2007 issue of the American Journal of Human Genetics. The authors are Min Shi, Kaare Christensen, Clarice R. Weinberg, Paul Romitti, Lise Bathum, Anthony Lozada, Richard W. Morris, Michael Lovett, and Jeffrey C.Murray.

The National Institute of Dental and Craniofacial Research (NIDCR) (http://www.nidcr.nih.gov) is the nation's leading funder of research on oral, dental, and craniofacial health.

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